Post-vasectomy sperm autoimmunogens in the Lewis rat

Vasectomy was performed on inbred Lewis rats to induce anti-sperm autoantibodies and to identify their cognate autoantigens. Different detergent extracts of cauda epididymal spermatozoa were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted to nitrocellulose, and probed with sera from pre-vasectomy, post-vasectomy, and hyperimmunized animals to detect isologous sperm antigens. Nonreduced SDS-soluble autoantigens at greater than 200, 86, 43, and 26 kDa were bound by post-vasectomy antisera. Reduction of SDS-soluble antigens resulted in increased staining of the 86, 72-63, and 43 kDa autoantigens. Laemmli extraction of SDS insoluble pellets with beta-mercaptoethanol generated the largest repertoire of autoantigens including several autoantigens found in SDS-soluble extracts. Therefore, to analyze the entire repertoire of post-vasectomy autoantigens, whole sperm were extracted with Laemmli buffer under reducing conditions. Autoantiserum from most vasectomized animals bound Laemmli-extracted reduced autoantigens of approximately 86 (89-78), 63, 43, and 20 (21-16) kDa. Testicular extracts, reduced and separated by SDS-PAGE, contained autoantigens of 76, 60, and 42 kDa that were also recognized by hyperimmune and post-vasectomy antisera. The repertoire of sperm antigens recognized by pooled serum from hyperimmunized animals was similar to the cumulative repertoire recognized by post-vasectomy sera. These studies define several major sperm autoimmunogens recognized by post-vasectomy antisera and indicate that many of these peptide autoimmunogens are disulfide-bonded complexes.     

Nucleotide sequences of accessible regions of 23S RNA in 50S ribosomal subunits.

Nucleotide sequences around kethoxal-reactive guanine residues of 23S RNA in 50S ribosomal subunits have been determined. By use of the diagonal paper electrophoresis method )Noller, H.F. (1974), Biochemistry 13, 4694-4703), 41 ribonuclease T1 oligonucleotides, originating from about 25 sites, were identified and sequenced. These sites are single stranded and accessible in free 50S subunits, and are thus potential sites for interaction with functional ligands during protein synthesis. Examination of these sequences for potential intermolecular base-pairing reveals the following: (1) There are 19 possible complementary combinations between exposed sequences in 16S and 23S RNA containing more than 4 base pairs: 15 containing 5 base pairs and 4 containing 6 base pairs. Nine of these complementary combinations contain 16S RNA sequences which we have previously shown to be protected from kethoxall by 50S subunits (Chapman, N.M., and Noller, H.F. (1977), J. Mol. Biol. 109, 131-149). (2) One of the exposed sites in 23S RNA has a sequence which is complementary to the invariant GT psi CR sequence in tRNA.     

Effect of testosterone on muscle mass and muscle protein synthesis

We have studied the effect of a pharmacological dose of testosterone enanthate (3 mg.kg-1.wk-1 for 12 wk) on muscle mass and total-body potassium and on whole-body and muscle protein synthesis in normal male subjects. Muscle mass estimated by creatinine excretion increased in all nine subjects (20% mean increase, P less than 0.02); total body potassium mass estimated by 40K counting increased in all subjects (12% mean increase, P less than 0.0001). In four subjects, a primed continuous infusion protocol with L-[1-13C]leucine was used to determine whole-body leucine flux and oxidation. Whole-body protein synthesis was estimated from nonoxidative flux. Muscle protein synthesis rate was determined by measuring [13C]leucine incorporation into muscle samples obtained by needle biopsy. Testosterone increased muscle protein synthesis in all subjects (27% mean increase, P less than 0.05). Leucine oxidation decreased slightly (17% mean decrease, P less than 0.01), but whole-body protein synthesis did not change significantly. Muscle morphometry showed no significant increase in muscle fiber diameter. These studies suggest that testosterone increases muscle mass by increasing muscle protein synthesis.     

Protein engineering of the propeptide of human factor IX

Vitamin-K-dependent plasma proteins contain a highly conserved propeptide sequence located between the classical hydrophobic leader sequence and the N-terminus of the mature protein. This acts as a recognition sequence for the vitamin-K-dependent carboxylase which catalyses the conversion of specific glutamate residues to gamma-carboxyglutamate (Gla) residues in the adjacent Gla domain. Protein engineering of the 18 residue propeptide from human factor IX has highlighted the importance of residues -16Phe and -10Ala with respect to carboxylase recognition. In addition, studies of haemophilia B patients have shown that C-terminal propeptide residues -4Arg and -1Arg are required for proteolysis of the propeptide from the mature protein. To extend these previous studies we have introduced two novel mutations into the propeptide of human factor IX at positions -17(Val----Asp) and -6(Leu----AsP), and studied the effect of these changes on gamma-carboxylation and proteolytic processing. Both mutations reduce the expression of a calcium-dependent epitope in the Gla domain; however, only -6Leu----Asp shows reduced binding to barium sulphate. In addition, this latter mutation prevents proteolytic processing of the propeptide. These data support the current hypothesis that the propeptide contains two recognition elements: one for carboxylase recognition located towards the N-terminus, and one for propeptidase recognition located near the C-terminus.     

JNK and NFκB dependence of apoptosis induced by vinblastine in human acute promyelocytic leukaemia cells

The relationship between the mitogen‐activated protein kinase response, nuclear factor‐κB (NFκB) expression and the apoptosis in human acute promyelocytic leukaemia NB4 cells treated with vinblastine was investigated in this work. Cell viability, subdiploid DNA and cell cycle were analysed by propidium iodide permeability and flow cytometry analyses. Apoptosis was determined by annexin V‐Fluorescein isothiocyanate assays. Western‐blot analysis was used for determination of expression levels of apoptotic factors (p53, Bax and Bcl2), intracellular kinases [serine/threonine‐specific protein kinase, extracellular signal‐regulated kinase and c‐Jun N‐terminal kinase (JNK)], NFκB factor and caspases. Electrophoretic mobility shift assay was usefully applied to study DNA‐NFκB interaction. In NB4 cells, vinblastine produces alteration of p53 and DNA fragmentation. Vinblastine treatment had an antiproliferative effect via the induction of apoptosis producing Bax/Bcl‐2 imbalance. Vinblastine treatment suppressed NFκB expression and depressed NFκB‐DNA binding activity while maintaining JNK activation that subsequently resulted in apoptotic response through caspase‐dependent pathway. Our study provides a possible anti‐cancer mechanism of vinblastine action on NB4 cells by deregulation of the intracellular signalling cascade affecting to JNK activation and NFκB expression. Moreover, JNK activation and NFκB depression can be very significant factors in apoptosis induction by vinblastine. Copyright © 2015 John Wiley & Sons, Ltd.     

Second harvest–Is there sufficient stubble for biofuel production in Australia?

Identifying the location and amount of grain crop residues (stubble) in Australia is necessary for determining the viability of potential biofuel plant locations. We combined 22 years of crop statistics with harvest indices and land use to arrive at spatially explicit stubble productivity figures. Stubble quantities using different focal radii and from different seasons provide an insight into the feasibility of its use for bioenergy. We focus on areas where the stubble concentrations within a 50 km radius were at least 500 kt per year; the amount suggested for a viable lignocellolosic bioethanol facility. The outcome of this study has been to show, for the first time, where there are large amounts of stubble in Australia. Whether the supply of stubble is sufficiently constant over time and indeed available at a price that is economic for a biofuel plant must be subject to future work.     

A NEW CLEARING-SQUASH TECHNIQUE FOR THE STUDY OF OVULE DEVELOPMENT IN ANGIOSPERMS

The simple, efficient method described here for the study of ovule and megagametophyte development in angiosperms provides for the extension of investigation beyond the limits imposed by the traditional but arduous section technique. Excised pistils previously fixed in FPA₅₀ and stored in 70 % ethanol are placed in a clearing fluid composed of lactic acid (85 %), chloral hydrate, phenol, clove oil, and xylene (2:2:2:2:1, by weight). After 24 hr, ovules dissected from the ovularies are transferred with some of the fluid to a slide, covered so that the cover glass is supported laterally by two permanently affixed covers, and examined with phase contrast optics. The unique action of the clearing fluid permits the study of cellular structure with the phase oil objective focused at any focal plane within the ovule. Downward focusing thus reveals a series of optical sections in the sagittal, frontal, or transverse plane depending on the orientation of the ovule. Orientation can be altered by a slight shifting of the cover glass on the lateral support mounts. The ovules become quite fragile in the clearing fluid. Pressure applied to the cover glass gradually breaks the ovule apart without disrupting the structural integrity of individual cells. This squash procedure provides for extending observations to cytological features of megasporocytes, megaspores, and megagametophytes previously identified in intact ovules. The new method is applied here to the study of ovule development in two unrelated species, Cassia abbreviata Oliver var. granitica Bak. f. (Leguminosae) and Ludwigia uruguayensis (Camb.) Hara. (Onagraceae). For best results, the ovules of Ludwigia must be pretreated in lactic acid (85 %) for 24 hr prior to application of the clearing fluid. Other methods for pretreatment likely will be required as the technique is applied to a wider range of flowering plant species.